FIG 8.

Confirmation of reduced gap junction plaque formation due to infection using Triton X-100 solubilization. Primary astrocytes were infected with MHV-A59 at an MOI of 2, and mock-infected cells were maintained in parallel. A membrane-enriched fraction was isolated from homogenized cells from each culture, and then protein was solubilized in the presence of 1% Triton X-100 at 4°C. Subsequently, these fractions were loaded in a gel and probed for Cx43. (A) Compared to levels for mock-infected controls, a reduction in both the total membrane fraction as well as the Triton X-100 insoluble fraction was observed upon MHV-A59 infection. (B) The amount of Cx43 present in the total membrane fraction was reduced 38.4% ± 6.9% in virus-infected cells compared to the level for mock-infected cells (means ± SEM; n = 3; *, P < 0.05). In mock-infected cultures, most of the Cx43 (81.6% ± 3.2%) was pooled in the Triton X-100 insoluble (Ins) fraction, whereas only 18.4% ± 3.3% was pooled in the soluble (Sol) fraction. In contrast, in MHV-A59-infected astrocytes (at an MOI of 2 at 24 h p.i.), large fractions of Cx43 (50.6% ± 2.5%) appeared in both the Triton X-100 soluble fraction and the Triton X-100 insoluble gap junction plaques (49.4% ± 2.5%). The insoluble versus soluble fraction ratio was calculated for mock- and virus-infected astrocytes. For virus-infected astrocytes this ratio (1.01) was significantly lower than that for mock-infected astrocytes (5.05; ***, P < 0.001).